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Interferons are important cytokines characterized by antiviral, antiproliferative, and immunomodulatory activities. These activities form a basis for the clinical benefits that have been observed in a number of diseases, including hepatitis, various cancers and multiple sclerosis. The interferons are divided into the type I and type II classes. Interferon xcex2 belongs to the class of type I interferons, which also includes interferons xcex1, xcfx84 and xcfx89, whereas interferon xcex3 is the only known member of the distinct type II class.
Human interferon xcex2 is a regulatory polypeptide with a molecular weight of 22 kDa consisting of 166 amino acid residues. It can be produced by most cells in the body, in particular fibroblasts, in response to viral infection or exposure to other biologics. It binds to a multimeric cell surface receptor, and productive receptor binding results in a cascade of intracellular events leading to the expression of interferon xcex2 inducible genes which in turn produces effects which can be classified as antiviral, antiproliferative and immunomodulatory.
The amino acid sequence of human interferon xcex2 was reported by Taniguchi, Gene 10:11-15, 1980, and in EP 83069, EP 41313 and U.S. Pat. No. 4,686,191.
Crystal structures have been reported for human and murine interferon xcex2, respectively (Proc. Natl. Acad. Sci. USA 94:11813-11818, 1997. J. Mol. Biol. 253:187-207, 1995). They have been reviewed in Cell Mol. Life Sci. 54:1203-1206, 1998.
Relatively few protein-engineered variants of interferon xcex2 have been reported (WO 9525170, WO 9848018, U.S. Pat. Nos. 5,545,723, 4,914,033, EP 260350, U.S. Pat. Nos. 4,588,585, 4,769,233, Stewart et al, DNA Vol 6 no2 1987 pp. 119-128, Runkel et al, 1998, Jour. Biol. Chem. 273, No. 14, pp. 8003-8008).
Expression of interferon xcex2 in CHO cells has been reported (U.S. Pat. Nos. 4,966,843, 5,376,567 and 5,795,779).
Redlich et al, Proc. Natl. Acad. Sci., USA, Vol. 88, pp. 4040-4044, 1991 disclose immunoreactivity of antibodies against synthetic peptides corresponding to peptide stretches of recombinant human interferon xcex2 with the mutation C17S.
Interferon xcex2 molecules with a particular glycosylation pattern and methods for their preparation have been reported (EP 287075 and EP 529300).
Various references disclose modification of polypeptides by polymer conjugation or glycosylation. Polymer modification of native interferon xcex2 or a C17S variant thereof has been reported (EP 229108, U.S. Pat. No. 5,382,657, EP 593868, U.S. Pat. No. 4,917,888 and WO 99/55377). U.S. Pat. No. 4,904,584 discloses PEGylated lysine depleted polypeptides, wherein at least one lysine residue has been deleted or replaced with any other amino acid residue. WO 99/67291 discloses a process for conjugating a protein with PEG, wherein at least one amino acid residue on the protein is deleted and the protein is contacted with PEG under conditions sufficient to achieve conjugation to the protein. WO 99/03887 discloses PEGylated variants of polypeptides belonging to the growth hormone superfamily, wherein a cysteine residue has been susbstituted with a non-essential amino acid residue located in a specified region of the polypeptide. Interferon xcex2 is mentioned as one example of a polypeptide belonging to the growth hormone superfamily. WO 00/23114 discloses glycosylated and pegylated interferon xcex2. WO 00/23472 discloses interferon xcex2 fusion proteins. WO 00/26354 discloses a method of producing a glycosylated polypeptide variant with reduced allergenicity, which as compared to a corresponding parent polypeptide comprises at least one additional glycosylation site. U.S. Pat. No. 5,218,092 discloses modification of granulocyte colony stimulating factor (G-CSF) and other polypeptides so as to introduce at least one additional carbohydrate chain as compared to the native polypeptide. Interferon xcex2 is mentioned as one example among many polypeptides which allegedly can be modified according to the technology described in U.S. Pat. No. 5,218,092.
Commercial preparations of interferon xcex2 are sold under the names Betaseron(copyright) (also termed interferon xcex21b, which is non-glycosylated, produced using recombinant bacterial cells, has a deletion of the N-terminal methionine residue and the C17S mutation), and Avonex(trademark) and Rebif(copyright) (also termed interferon xcex21a, which is glycosylated, produced using recombinant mammalian cells) for treatment of patients with multiple sclerosis, and have been shown to be effective in reducing the exacerbation rate. More patients treated with these interferon xcex2 agents remain exacerbation-free for prolonged periods of time as compared with placebo-treated patients. Furthermore, the accumulation rate of disability is reduced (Neurol. 51:682-689, 1998).
Comparison of interferon xcex21a and xcex21b with respect to structure and function has been presented in Pharmaceut. Res. 15:641-649, 1998.
Interferon xcex2 is the first therapeutic intervention shown to delay the progression of multiple sclerosis, a relapsing then progressive inflammatory degenerative disease of the central nervous system.
Its mechanism of action, however, remains largely unclear. It appears that interferon xcex2 has inhibitory effects on the proliferation of leukocytes and antigen presentation. Furthermore, interferon xcex2 may modulate the profile of cytokine production towards an anti-inflammatory phenotype. Finally, interferon xcex2 can reduce T-cell migration by inhibiting the activity of T-cell matrix metalloproteases. These activities are likely to act in concert to account for the mechanism of interferon xcex2 in MS (Neurol. 51:682-689, 1998).
In addition, interferon xcex2 may be used for the treatment of osteosarcoma, basal cell carcinoma, cervical dysplasia, glioma, acute myeloid leukemia, multiple myeloma, Hodgkin""s disease, breast carcinoma, melanoma, and viral infections such as papilloma virus, viral hepatitis, herpes genitalis, herpes zoster, herpetic keratitis, herpes simplex, viral encephalitis, cytomegalovirus pneumonia, and rhinovirus.
Various side effects are associated with the use of current preparations of interferon xcex2, including injection site reactions, fever, chills, myalgias, arthralgias, and other flu-like symptoms (Clin. Therapeutics, 19:883-893, 1997).
In addition, 6-40% of patients develop neutralizing antibodies to interferon xcex2 (Int. Arch. Allergy Immunol. 118:368-371, 1999). It has been shown that development of interferon xcex2-neutralizing antibodies decreases the biological response to interferon xcex2, and cause a trend towards decreased treatment efficacy (Neurol. 50:1266-1272, 1998). Neutralizing antibodies will likely also impede the therapeutic utility of interferon xcex2 in connection with treatment of other diseases (Immunol. Immuther. 39:263-268, 1994).
Given the magnitude of side effects with current interferon xcex2 products, their association with frequent injection, the risk of developing neutralizing antibodies impeding the desired therapeutic effect of interferon xcex2, and the potential for obtaining more optimal therapeutic interferon xcex2 levels with concomitant enhanced therapeutic effect, there is clearly a need for improved interferon xcex2-like molecules.
This application discloses improved interferon xcex2 molecules providing one or more of the aforementioned desired benefits. In particular conjugates are disclosed that exhibit interferon xcex2 activity and comprise at least one non-polypeptide moiety covalently attached to an interferon xcex2 polypeptide that comprises an amino acid sequence that differs from that of wild-type human interferon xcex2 with the amino acid sequence shown in SEQ ID NO 2 in at least one amino acid residue selected from an introduced or removed amino acid residue comprising an attachment group for the non-polypeptide moiety. Conjugates of the present invention have a number of improved properties as compared to human interferon xcex2, including reduced immunogenicity, increased functional in vivo half-life, increased serum half-life, and/or increased bioavailability. Consequently, the conjugate of the invention offers a number of advantages over the currently available interferon xcex2 compounds, including longer duration between injections, fewer side effects, and/or increased efficiency due to reduction in antibodies. Moreover, higher doses of active protein and thus a more effective therapeutic response may be obtained by use of a conjugate of the invention. Furthermore, conjugates of the invention have demonstrated significantly reduced cross-reactivity with sera from patients treated with currently available interferon xcex2 products as defined hereinbelow.
In one aspect the invention relates to a conjugate exhibiting interferon xcex2 activity and comprising at least one first non-polypeptide moiety covalently attached to an interferon xcex2 polypeptide, the amino acid sequence of which differs from that of wild-type human interferon xcex2 in at least one introduced and at least one removed amino acid residue comprising an attachment group for said first non-polypeptide moiety.
In another aspect the invention relates to a conjugate exhibiting interferon xcex2 activity and comprising at least one first non-polypeptide moiety conjugated to at least one lysine residue of an interferon xcex2 polypeptide, the amino acid sequence of which differs from that of wild-type human interferon xcex2 in at least one introduced and/or at least one removed lysine residue.
In yet another aspect the invention relates to a conjugate exhibiting interferon xcex2 activity and comprising at least one first non-polypeptide moiety conjugated to at least one cysteine residue of an interferon xcex2 polypeptide, the amino acid sequence of which differs from at least one introduce cysteine residue into a position that in wild-type human interferon xcex2 is occupied by a surface exposed amino acid residue.
In yet another aspect the invention relates to a conjugate exhibiting interferon xcex2 activity and comprising at least one first non-polypeptide moiety having an acid group as an attachment group, which moiety is conjugated to at least one aspartic acid or glutamic acid residue of an interferon xcex2 polypeptide, the amino acid sequence of which differs from that of wild-type human interferon xcex2 in at least one introduced and/or at least one removed aspartic acid or glutamic acid residue.
In yet another aspect the invention relates to a conjugate exhibiting interferon xcex2 activity and comprising at least one polymer molecule and at least one sugar moiety covalently attached to an interferon xcex2 polypeptide, the amino acid sequence of which differs from that of wild-type human interferon xcex2 in
(a) at least one introduced and/or at least one removed amino acid residue comprising an attachment group for the polymer molecule, and
(b) at least one introduced and/or at least one removed amino acid residue comprising an attachment group for the sugar moiety,
provided that when the attachment group for the polymer molecule is a cysteine residue, and the sugar moiety is an N-linked sugar moiety, a cysteine residue is not inserted in such a manner that an N-glycosylation site is destroyed.
In yet another aspect the invention relates to a conjugate exhibiting interferon xcex2 activity and comprising an interferon xcex2 polypeptide, the amino acid sequence of which differs from that of wild-type human interferon xcex2 in at least one introduced glycosylation site, the conjugate further comprising at least one un-PEGylated sugar moiety attached to an introduced glycosylation site.
In yet another aspect the invention relates to a conjugate exhibiting interferon xcex2 activity and comprising an interferon xcex2 polypeptide, the amino acid sequence of which differs from that of wild-type human interferon xcex2 in that a glycosylation site has been introduced or removed by way of introduction or removal of amino acid residue(s) constituting a part of a glycosylation site in a position that in wildtype human interferon xcex2 is occupied by a surface exposed amino acid residue.
In a still further aspect the invention relates to a conjugate exhibiting interferon xcex2 activity and comprising a sugar moiety covalently attached to an interferon xcex2 polypeptide, the amino acid sequence of which differs from that of wild-type human interferon xcex2 in at least one removed glycosylation site.
In still further aspects the invention relates to means and methods for preparing a conjugate or interferon xcex2 polypeptide for use in the invention, including nucleotide sequences and expression vectors encoding the polypeptide as well as methods for preparing the polypeptide or the conjugate.
In final aspects the invention relates to a therapeutic composition comprising a conjugate of the invention, to a conjugate or composition of the invention for use in therapy, to the use of a conjugate or composition in therapy or for the manufacture of a medicament for treatment of diseases.